New Zosteropenillines and Pallidopenillines from the Seagrass-Derived Fungus Penicillium yezoense KMM 4679

Ten new decalin polyketides, zosteropenilline M (1), 11-epi-8-hydroxyzosteropenilline M (2), zosteropenilline N (3), 8-hydroxyzosteropenilline G (4), zosteropenilline O (5), zosteropenilline P (6), zosteropenilline Q (7), 13-dehydroxypallidopenilline A (8), zosteropenilline R (9) and zosteropenilline S (10), together with known zosteropenillines G (11) and J (12), pallidopenilline A (13) and 1-acetylpallidopenilline A (14), were isolated from the ethyl acetate extract of the fungus Penicillium yezoense KMM 4679 associated with the seagrass Zostera marina. The structures of isolated compounds were established based on spectroscopic methods. The absolute configurations of zosteropenilline Q (7) and zosteropenilline S (10) were determined using a combination of the modified Mosher’s method and ROESY data. The absolute configurations of zosteropenilline M (1) and zosteropenilline N (3) were determined using time-dependent density functional theory (TD-DFT) calculations of the ECD spectra. A biogenetic pathway for compounds 1–14 is proposed. The antimicrobial, cytotoxic and cytoprotective activities of the isolated compounds were also studied. The significant cytoprotective effects of the new zosteropenilline M and zosteropenillines O and R were found in a cobalt chloride (II) mimic in in vitro hypoxia in HEK-293 cells. 1-Acetylpallidopenilline A (14) exhibited high inhibition of human breast cancer MCF-7 cell colony formation with IC50 of 0.66 µM and its anticancer effect was reduced when MCF-7 cells were pretreated with 4-hydroxitamoxifen. Thus, we propose 1-acetylpallidopenilline A as a new xenoestrogen with significant activity against breast cancer.


Introduction
Marine-derived fungi still hold a leading position in terms of the number of newly isolated compounds each year [1].Penicillium yezoense belongs to the Aspergilloides section and the Thomiorum series, which includes the 12 species, P. fusisporum, P. aurantioviolaceum, P. valentinum, P. roseoviride, P. cartierense, P. thomii, P. yezoense, P. contaminatum, P. austroafricanum, P. crocicola, P. grevilleicola and P. jejuense.Earlier, P. yezoense was treated as a synonym of P. thomii, and then they were accepted as distinct species [2].Six species in this series were described relatively recently, in 2014-2015, including P. jejuense, which was found in marine habitats [2,3].Representatives of the Thomiorum series in terrestrial environments are most often associated with substrates of plant origin and are also found

Molecular Identification of the Fungal Strain/Identification of Penicillium yezoense КММ 4679
The strain KMM 4679 was identified using molecular markers such as the ITS region and partial BenA, CaM and RPB2 genes.Approximately 600 bp fragments of the ITS region, about 500 bp fragments of the partial BenA gene, about 600 bp fragments of the partial CaM gene and about 1200 bp fragments of the partial RPB2 gene were amplified and sequenced.According to the results of BLAST analysis, the sequence of the ITS region was 100% identical to the sequence of the ex-type strain Penicillium yezoense CBS 350.59T, while the BenA and RPB2 gene sequences were more than 99% identical, and the CaM gene sequence was more than 98% identical.The phylogenetic Maximum Likelihood (ML) tree of the concatenated ITS-BenA-CaM-RPB2 sequences clearly showed that the strain

Structural Elucidation
The molecular formula of 1 was established to be C15H24O3 by the HRESIMS peak at m/z 275.1611 [M + Na] + (Figure S1) and 13 C NMR analyses.A close inspection of 1 H and 13 C NMR, DEPT and HSQC data for 1 (Tables 1 and 2) revealed the presence of two methyl (δH 0.92, δC 22.The relative configurations of 1 were assigned based on a ROESY experiment and 1 H-1 H coupling constants (Table 2).Observed ROESY correlations between H-4 and H-10, between H-5 and H-11, H a -2, and the magnitudes of the vicinal coupling constants H-4 Mar. Drugs 2024, 22, 317 5 of 25 (J = 10.0 Hz), H-5 (J = 11.5, 4.0 Hz) and H-11 (J = 9.0 Hz), indicated a trans-fusion of the A and B rings, an α-orientation of the 11-OH group and a β-orientation of the side chain at C-4.The orientation of H 3 -15 was determined to be β based on key ROESY correlations between H-8 and H-10 and biogenetic relationships with previously isolated polyketides [17,18].The absolute stereochemistry of 1 was established based on quantum chemical calculations of the ECD spectrum.The conformational analysis of 1 was performed at the B3LYP/6-31G(d)_PCM level of theory.The internal rotations of the substituents at C-4 and C-11, as well as of the alkyl substituent at C-3, were considered.We found that the rotameric forms of 1 containing intramolecular hydrogen bonds were the most stable conformations.All other conformations lost to them by more than 3 kcal/mol (Figure S114).Conformations with a dihedral angle θ1 = ∠O−C(3)−C(4) −H(4) ≈ 180 • were the most stable (Figure S114), and ECD spectra corresponding to them contained a very intensive positive band at λ ≈ 210 nm.The minor conformations, in which a carbonyl group stayed with a dihedral angle θ1 ≈ 0 • , generated ECD spectra, in which the bands at λ ≈ 210 nm and at λ ≈ 290 nm had similar intensities (Figure S115).The experimental ECD spectrum of 1 contained two positive bands in the region λ ≥ 200 nm. Figure 4 presents a comparison of this spectrum with the calculated statistically averaged spectrum for 4R,5S,8S,10S,11S-1.A comparison of the features of the calculated and experimental ECD spectra showed good qualitative mutual correspondence.Thus, the absolute configuration of 1 was established to be 4R,5S,8S,10S,11S.Compound 1 was named zosteropenilline M. It is worth noting that a C-4-methylated derivative of zosteropenilline M (1) viridicatumone A was reported from halotolerant plant-associated Penicillium viridicatum [26].
Mar. Drugs 2024, 22, x 5 of 27 The relative configurations of 1 were assigned based on a ROESY experiment and 1 H-1 H coupling constants (Table 2).Observed ROESY correlations between H-4 and H-10, between H-5 and H-11, Ha-2, and the magnitudes of the vicinal coupling constants H-4 (J = 10.0 Hz), H-5 (J = 11.5, 4.0 Hz) and H-11 (J = 9.0 Hz), indicated a trans-fusion of the A and B rings, an α-orientation of the 11-OH group and a β-orientation of the side chain at C-4.The orientation of H3-15 was determined to be β based on key ROESY correlations between H-8 and H-10 and biogenetic relationships with previously isolated polyketides [17,18].The absolute stereochemistry of 1 was established based on quantum chemical calculations of the ECD spectrum.The conformational analysis of 1 was performed at the B3LYP/6-31G(d)_PCM level of theory.The internal rotations of the substituents at C-4 and C-11, as well as of the alkyl substituent at C-3, were considered.We found that the rotameric forms of 1 containing intramolecular hydrogen bonds were the most stable conformations.All other conformations lost to them by more than 3 kcal/mol (Figure S114).Conformations with a dihedral angle 1 = O−C(3)−C(4) −H(4)  180 were the most stable (Figure S114), and ECD spectra corresponding to them contained a very intensive positive band at   210 nm.The minor conformations, in which a carbonyl group stayed with a dihedral angle 1  0, generated ECD spectra, in which the bands at   210 nm and at   290 nm had similar intensities (Figure S115).The experimental ECD spectrum of 1 contained two positive bands in the region   200 nm. Figure 4 presents a comparison of this spectrum with the calculated statistically averaged spectrum for 4R,5S,8S,10S,11S-1.A comparison of the features of the calculated and experimental ECD spectra showed good qualitative mutual correspondence.Thus, the absolute configuration of 1 was established to be 4R,5S,8S,10S,11S.Compound 1 was named zosteropenilline M. It is worth noting that a C-4-methylated derivative of zosteropenilline M (1) viridicatumone A was reported from halotolerant plant-associated Penicillium viridicatum [26].    1 and 2) observed for 2 were similar to those observed for 1. Analysis of the proton and carbon signals in the 1 H and 13 C NMR spectra of 2, including DEPT and HSQC experiments (Tables 1 and 2), revealed the presence of two methyl (δ H 1.  S119) established the structure of the A ring of 2 including the location of hydroxy and methyl groups at C-8. Observed magnitude of the 3 J coupling constant ( 3 J H5-H10 = 12.0 Hz) indicated trans-fusion of the A and B rings, 3 J coupling constant ( 3 J H4-H5 = 9.9 Hz) indicated β-orientation of the side chain at C-4, and 3 J H10-H11 (3.3 Hz) indicated β-equatorial orientation of 11-OH group in 2. The β-orientation C-15 was confirmed by the ROESY correlation between H-5 and H 3 -15.Thus, compound 2 was named 11-epi-8-hydroxyzosteropenilline M.
The molecular formula of 3 was established to be C 14 H 22 O 3 by an HRESIMS peak at m/z 261.1473 [M + Na] + (Figure S17) and corresponded with 13 C NMR data.The chemical shift values of B ring atoms in the 1 H and 13 C NMR spectra of 3 (Tables 1 and 2) were very similar to those obtained for compound 2. A close inspection of 1 H and 13  The absolute configurations of the stereocenters of 3 were established using calculated ECD data (Figure 5).The experimental ECD spectrum of 3 contained an intense negative band in the λ ≤ 207 nm region and two positive bands in the λ ≥ 207 nm region.The band at λ ≈ 290 nm was more intense than that at λ ≈ 212 nm.A comparison of ECD spectra calculated for different conformations of 3 showed that the sign and position of the band at λ ≈ 290 nm were the same for all selected conformations.On the contrary, the shape of the calculated ECD spectrum in the λ ≤ 264 nm region was very conformationally dependent (Figure S116).The shape of the final statistically averaged ECD spectrum depends hardly on the values of calculated statistical weights, which are themselves dependent on the chosen level of theoretical modeling.For 3, this is a possible cause for why the theoretical ECD spectrum deviates from the experimental one in the λ ≤ 275 nm region (Figure 5).An ECD spectrum of 4R,5S,8S,10S,11R-3 was modeled for situations, when total amounts of both types of conformations (dihedral angle 0 • :180 • ) related as 40:60, 60:40 and 80:20 (Figure S117).It is seen that the growth of the number of conformations with a dihedral angle 0 • leads to better qualitative correspondence of calculated and experimental ECD spectra.Thus, the absolute stereochemistry of 3 was determined to be 4R,5S,8S,10S,11R.Compound 3 was named zosteropenilline N.
The molecular formula of 4 was established to be C 15 H 24 O 4 by the HRESIMS peak at m/z 291.1579 [M + Na] + (Figure S25), which was confirmed by 13 C NMR analyses.The 1 H and 13 C NMR spectra (Tables 1 and 2) for compound 4 were very similar to those obtained for zosteropenilline G (11), previously isolated from the Penicillium thomii KMM 4674 [18], except for the C-7-C-10 and C-15 carbon signals and peaurantiogriseol D previously isolated from the Penicillium aurantiogriseum [27], except the lacking methyl at C-4. ROESY correlation of H 3 -14 (δ H 1.20)/H-5 (δ H 1.50) confirmed the β-orientation of the methyl group at C-13.Moreover, the ROESY correlation H-4 (δ H 2.95)/H-10 (δ H 2.31), together with the observed magnitude of the vicinal coupling constant H-4 (J = 11.6 Hz), confirmed the trans-fusion of the A and B rings and the β-orientation of the 3-hydroxy-1-oxopropyl residue at C-4.Thus, compound 4 was named 8-hydroxyzosteropenilline G.
The molecular formula of 5 was established to be C 14 H 22 O 3 by the HRESIMS peak at m/z 261,1471 [M + Na] + (Figure S32) and was confirmed by 13 C NMR analyses.The 1 H and 13 C NMR data (Tables 1 and 2) observed for 5 closely resembled those obtained for 4, except for signals of C-1 and C-2.A close inspection of the 1 H and 13 C NMR, DEPT and HSQC data of 5 revealed that the A and B rings were the same as in 4, and the acetyl group at C-4 was the same as in 3. ROESY correlation H 3 -14 (δ H 1.19)/H-5 (δ H 1.47) confirmed the β-orientation H 3 -14.Thus, the structure of compound 5 was established and named zosteropenilline O.This compound is very similar to libertalide C, which has an additional methyl group at C-4 and was isolated from the coral-derived fungus Libertasomyces sp.[28].
hardly on the values of calculated statistical weights, which are themselves dependent on the chosen level of theoretical modeling.For 3, this is a possible cause for why the theoretical ECD spectrum deviates from the experimental one in the   275 nm region (Figure 5).An ECD spectrum of 4R,5S,8S,10S,11R-3 was modeled for situations, when total amounts of both types of conformations (dihedral angle 0:180) related as 40:60, 60:40 and 80:20 (Figure S117).It is seen that the growth of the number of conformations with a dihedral angle 0 leads to better qualitative correspondence of calculated and experimental ECD spectra.Thus, the absolute stereochemistry of 3 was determined to be 4R,5S,8S,10S,11R.Compound 3 was named zosteropenilline N.   3 and 4) revealed the presence of two methyl groups (δ H 1.18, δ C 25. ).These data and four degrees of unsaturation from the molecular formula suggested that compound 6 possessed two rings, one double bond and one keto group. 1 H-1 H COSY data and HMBC correlations (Figure S119) revealed the presence of a decalin moiety and established a ∆ 11 double bond, the location of a methyl group at C-13, the location of an oxygen-bearing methylene group at C-8 and the location of an acetyl group at C-4 in 6. ROESY correlation H-5 (δ H 1.49)/H 3 -14 (δ H 1.18) and H 2 -15 (δ H 3.48, 3.45) confirmed the β-orientation methyl and methylene groups at C-13 and C-8, accordingly.Thus, compound 6 was named zosteropenilline P. The molecular formula of 7 was established to be C 14 H 22 O 3 based on the HRESIMS peak at m/z 261.1469 [M + Na] + (Figure S48).The 1 H and 13 C NMR spectra for  S119) established the structure of the A ring of 7 including the location of hydroxyl and methyl groups at C-9 and C-8, respectively.The ROESY correlation H-5 (δ H 1.59)/H 3 -14/H-9 together with observed magnitudes of the vicinal coupling constant of H-9 (J = 9.8 Hz) established the α-configuration of the 9-OH group and β-orientation H 3 -14.The absolute configuration of C-9 was determined using the modified Mosher's method [29].Esterification of 7 with (R)-and (S)-MTPA chlorides occurred at the C-9 hydroxy group to give (S)-and (R)-MTPA esters 7b and 7a, respectively.The observed chemical shift differences ∆δ (δ S − δ R ) (Figure 6) indicated a 9R configuration.These data and the observed magnitudes of the vicinal coupling constants H-4 (δ H 2.88, J = 11.7 Hz), H-9 (δ H 2.83, J = 9.8 Hz) and H-10 (δ H 1.80, J = 10.0, 2.2 Hz) determined the absolute stereostructure of 7 as 4R,5S,8S,9R,10R,13R, which was the same as that of pallidopenilline A [17].Thus, compound 7 was named zosteropenilline Q.The molecular formula of 8 was established to be C 15 H 24 O 3 based on the HRESIMS peak at m/z 275.1592 [M + Na] + (Figure S56).Analysis of 1 H and 13 C NMR, DEPT and HSQC data of 8 (Tables 3 and 4) revealed the presence of a decalin moiety, as well as an established ∆ 11 double bond, the location of methyl groups at C-8 and C-13, the location of The planar structure of compound 8 was similar to pallidopenilline A [17], except for the absence of 13-ОН, and close to craterellone B [30], except for the 3-hydroxy-1-oxopropyl residue at C-4 instead of an acetyl group and the absence of the methyl group at C-4.The observed magnitudes of the vicinal coupling constants of H-4 (J = 11.2, 6.0 Hz) and H-13 (J = 6.0, 4.0 Hz) established the β-configuration of the methyl group at C-13.Thus, compound 8 was named 13-dehydroxypallidopenilline A.
The molecular formula of 9 was established to be C 15 H 24 O 4 based on the HRESIMS peak at m/z 291.1579 [M + Na] + (Figure S64).The 1 H and 13 C NMR spectra (Tables 3 and 4) for compound 9 were very similar to those obtained for zosteropenilline L [18]  The molecular formula of 8 was established to be C15H24O3 based on the HRESIMS peak at m/z 275.1592 [M + Na] + (Figure S56).Analysis of 1 H and 13 C NMR, DEPT and HSQC data of 8 (Tables 3 and 4) revealed the presence of a decalin moiety, as well as an established Δ 11 double bond, the location of methyl groups at C-8 and C-13, the location of a hydroxy group at C-9, and the location of a 3-hydroxy-1-oxopropyl residue at C-4 in 8.The planar structure of compound 8 was similar to pallidopenilline А [17], except for the absence of 13-ОН, and close to craterellone B [30], except for the 3-hydroxy-1-oxopropyl residue at C-4 instead of an acetyl group and the absence of the methyl group at C-4.The observed magnitudes of the vicinal coupling constants of H-4 (J = 11.2, 6.0 Hz) and H-13 (J = 6.0, 4.0 Hz) established the β-configuration of the methyl group at C-13.Thus, compound 8 was named 13-dehydroxypallidopenilline A.
The molecular formula of 9 was established to be C15H24O4 based on the HRESIMS peak at m/z 291.1579 [M + Na] + (Figure S64).The 1 H and 13 C NMR spectra (Tables 3 and 4) for compound 9 were very similar to those obtained for zosteropenilline L [18]   The absolute configuration of C-12 was determined using the modified Mosher's method [29].Esterification of 9 with (R)-and (S)-MTPA chlorides occurred at the C-12 hydroxy group to give the (S)-and (R)-MTPA esters 9b and 9a, respectively.The observed chemical shift differences ∆δ (δS − δR) (Figure 8A) indicated the 12R configuration.Thus, the full absolute stereostructure of 9 was determined as 4R,5R,8S,12R,13S.Compound 9 was named zosteropenilline R. It should be noted that earlier, the same configuration of C-12 was reported for zosteropenilline L [18], while chemical shift values of C-12 and H-12 were very different in comparison with those for 9.It is possible that the NOESY data were incorrectly interpreted when determining the stereochemistry of zosteropenilline L. The absolute configuration of C-12 was determined using the modified Mosher's method [29].Esterification of 9 with (R)-and (S)-MTPA chlorides occurred at the C-12 hydroxy group to give the (S)-and (R)-MTPA esters 9b and 9a, respectively.The observed chemical shift differences ∆δ (δ S − δ R ) (Figure 8A) indicated the 12R configuration.Thus, the full absolute stereostructure of 9 was determined as 4R,5R,8S,12R,13S.Compound 9 was named zosteropenilline R. It should be noted that earlier, the same configuration of C-12 was reported for zosteropenilline L [18], while chemical shift values of C-12 and H-12 were very different in comparison with those for 9.It is possible that the NOESY data were incorrectly interpreted when determining the stereochemistry of zosteropenilline L.

Biological Activity of Isolated Compounds 2.3.1. Antimicrobial Activity
The effects of compounds 1, 2 and 4-14 on Staphylococcus aureus ATCC 21027, Escherichia coli VKPM (B-7935) and Candida albicans KMM 455 test strain growth and biofilm formation were investigated, and the data are presented in Table 5. Compound 3 was isolated in insufficient amounts and was not investigated in all experiments.
Thus, the antimicrobial activities of the isolated compounds were weak.None of the tested compounds exhibited antimicrobial activity against E. coli.Compounds 1 and 6-14 at 100 µM inhibited the growth of S. aureus by nearly 15-30%.Compounds 1, 2, 4-7, 9, 11 and 12 inhibited the growth of C. albicans by 6-35% at a concentration of 100 µM.Nevertheless, none of the studied compounds affected the formation of biofilms of the test strains of microorganisms at concentrations up to 100 µM.

Cytotoxic Activity of Isolated Compounds
The influence of compounds 1, 2 and 4-14 on the viability of normal human kidney HEK 298 cells, as well as human prostate PC-3, cervical HeLa and breast MCF-7 cancer cells, was measured using the MTT assay after 24 h of treatment.The results are presented in Table 6.None of the investigated compounds showed a significant cytotoxic effect on normal kidney cells after 24 h.Compounds 5−7, only at a concentration of 100 µM, decreased the viability of HEK-298 cells by approximately 15-20%.The cytotoxic effect of the compounds on PC-3 cell viability was weak, excluding that of compound 14.The concentration of half-maximal inhibition (IC 50 ) for 14 was calculated as 94.20 ± 1.13 µM.The effect of these compounds on HeLa cells was more pronounced.The IC 50s values calculated for 2, 5 and 11 were 87.29, 82.49 and 79.68 µM, respectively.Moreover, compound 14 half-maximally inhibited the viability of MCF-7 cells at 71.98 ± 2.48 µM.

Cytoprotective Activity
The cytoprotective effects of the isolated compounds were investigated in cobalt (II) chloride-induced HEK-293 cells, and some compounds showed significant effects in this in vitro model.The influence of compounds 1, 5, 9 and 10 on the viability and lipid peroxidation levels in CoCl 2 -treated cells is presented in Figure 9.
of these compounds on HeLa cells was more pronounced.The IC50s values calculated for 2, 5 and 11 were 87.29, 82.49 and 79.68 µ M, respectively.Moreover, compound 14 halfmaximally inhibited the viability of MCF-7 cells at 71.98 ± 2.48 µ M.

Cytoprotective Activity
The cytoprotective effects of the isolated compounds were investigated in cobalt (II) chloride-induced HEK-293 cells, and some compounds showed significant effects in this in vitro model.The influence of compounds 1, 5, 9 and 10 on the viability and lipid peroxidation levels in CoCl2-treated cells is presented in Figure 9.

Anticancer Activity of 1-Acetylpallidopenilline A (14)
In the experiments (Section 2.3.2),only 1-acetylpallidopenilline A ( 14) showed some cytotoxic activity against cancer PC-3, HeLa and MCF-7 cells compared to normal HEK-293 cells.Therefore, various aspects of the anticancer activity of compound 14 were investigated.
The exposition of PC-3, HeLa and MCF-7 cells with 14 for 48 h and 72 h resulted in an increase in its cytotoxicity (Figure 10).The IC50s values (for PC-3 cells) were calculated as 63.35  The toxic effect of CoCl 2 resulted in a significant increase in lipid peroxide levels in HEK-293 cells, which was measured using the MitoCLox fluorescence dye.Compounds 1, 5 and 9 significantly decreased lipid peroxide levels in CoCl 2 -treated HEK-293 cells (Figure 9B).

Anticancer Activity of 1-Acetylpallidopenilline A (14)
In the experiments (Section 2.3.2),only 1-acetylpallidopenilline A ( 14) showed some cytotoxic activity against cancer PC-3, HeLa and MCF-7 cells compared to normal HEK-293 cells.Therefore, various aspects of the anticancer activity of compound 14 were investigated.
The exposition of PC-3, HeLa and MCF-7 cells with 14 for 48 h and 72 h resulted in an increase in its cytotoxicity (Figure 10).The IC 50s values (for PC-3 cells) were calculated as 63.35 µM and 48.48 µM, respectively.The IC 50s values (for HeLa cells) were calculated as 82.71 µM and 73.15 µM, respectively.Finally, The IC 50s values (for MCF-7 cells) were calculated as 54.72 µM and 41.03 µM, respectively.The effects of 14 on PC-3, HeLa and MCF-7 cell colony formation were investigated, and the data are presented in Figure 11.The effects of 14 on PC-3, HeLa and MCF-7 cell colony formation were investigated, and the data are presented in Figure 11.The effects of 14 on PC-3, HeLa and MCF-7 cell colony formation were investigated, and the data are presented in Figure 11.The effect of 14 on colony formation was very significant.The half-maximal concentrations of the inhibition of PC-3, HeLa and MCF-7 colony formation was calculated for 14 as 2.48 ± 0.11 µ M, 0.96 ± 0.02 µ M and 0.66 ± 0.01 µ M, respectively.
The data on the effect of 14 on PC-3, HeLa and MCF-7 cell migration in the scratch assay are presented in Figure 12.The effect of 14 on colony formation was very significant.The half-maximal concentrations of the inhibition of PC-3, HeLa and MCF-7 colony formation was calculated for 14 as 2.48 ± 0.11 µM, 0.96 ± 0.02 µM and 0.66 ± 0.01 µM, respectively.
The data on the effect of 14 on PC-3, HeLa and MCF-7 cell migration in the scratch assay are presented in Figure 12.Thus, 14 significantly inhibited the migration of HeLa and MCF-7 cells (Figure 12), but did not affect the migration of PC-3 cells.
Finally, we investigated the combined cytotoxic effects of doxorubicin and 14 added together on the viability of PC-3, HeLa and MCF-7 cells.The data are presented in Figure 13.Thus, 14 significantly inhibited the migration of HeLa and MCF-7 cells (Figure 12), but did not affect the migration of PC-3 cells.Finally, we investigated the combined cytotoxic effects of doxorubicin and 14 added together on the viability of PC-3, HeLa and MCF-7 cells.The data are presented in Figure 13.Thus, 14 significantly inhibited the migration of HeLa and MCF-7 cells (Figure 12), but did not affect the migration of PC-3 cells.
Finally, we investigated the combined cytotoxic effects of doxorubicin and 14 added together on the viability of PC-3, HeLa and MCF-7 cells.The data are presented in Figure 13.Therefore, doxorubicin at a concentration of 1 µ M decreased MCF-7 cell viability by approximately 60%, and the combination of compound 14 with doxorubicin was more toxic.A more significant effect was observed when doxorubicin administered at 0.1 µ M. It decreased MCF-7 viability by only 30%, but the combination of 14 with doxorubicin diminished MCF-7 viability by 50%.
Thus, the more significant activity of 14 against MCF-7 cells, and to a lesser extent against HeLa cells, in contrast to PC-3, was observed in various experiments.To detect the possible target of 14, we used 4-hydroxytamoxifen (4-OHT) at a concentration of 10 Therefore, doxorubicin at a concentration of 1 µM decreased MCF-7 cell viability by approximately 60%, and the combination of compound 14 with doxorubicin was more toxic.A more significant effect was observed when doxorubicin was administered at 0.1 µM.It decreased MCF-7 viability by only 30%, but the combination of 14 with doxorubicin diminished MCF-7 viability by 50%.
Thus, the more significant activity of 14 against MCF-7 cells, and to a lesser extent against HeLa cells, in contrast to PC-3, was observed in various experiments.To detect the possible target of 14, we used 4-hydroxytamoxifen (4-OHT) at a concentration of 10 µM as a known inhibitor of estrogen receptors (ERs), and the data are presented in Figure 14.µ M as a known inhibitor of estrogen receptors (ERs), and the data are presented in Figure 14.No significant differences were detected between 14 and 14 after pretreatment with 4-OHT in HeLa and PC-3 cells.However, when MCF-7 cells were pretreated with 4-OHT and then treated with 14, a significant decrease in the cytotoxicity of 14 was observed.This confirmed that ERs are one of the possible targets for this fungal metabolite.

Discussion
Despite the relative occurrence of polyketides similar to isolated compounds 1-14 No significant differences were detected between 14 and 14 after pretreatment with 4-OHT in HeLa and PC-3 cells.However, when MCF-7 cells were pretreated with 4-OHT and then treated with 14, a significant decrease in the cytotoxicity of 14 was observed.This confirmed that ERs are one of the possible targets for this fungal metabolite.

Discussion
Despite the relative occurrence of polyketides similar to isolated compounds 1-14 [28,30], the absence of a methyl group at C-3 is a characteristic feature of only two related series of heptaketides: zosteropenillines and pallidopenillines, produced by several strains of Penicillium spp.[17,18].The biosynthesis of such heptaketides has not been reported before, but it is obvious that it occurs similarly to the biosynthesis of the nonaketide derivative lovastatin, which has a very similar decalin moiety [31].Thus, the decalin framework is formed as a result of the intramolecular Diels-Alder cyclization of the linear heptaketide precursor.It is likely that the C-9 hydroxy derivative 8 and the C-13 hydroxy derivative zosteropenilline G (11) are obtained directly from the formed intermediate i-5 (Figure 15A).According to our suggestion, zosteropenilline G ( 11) is a key intermediate for most other compounds.Compounds 4, 12 and 13 are simple hydroxy derivatives of zosteropenilline G (11).Compounds 5-7 with shortened side chains are possible products of the oxidative decarboxylation of the corresponding compounds 4, 12 and 13 with a "normal" side chain.The action of dioxygenase on the double bond of zosteropenilline G (11) with the subsequent hydrolysis of peroxides can lead to the formation of β-cisdiol i-7 and α-cis-diol i-10 (Figure 15B).The dehydration of the latter probably leads to the formation of zosteropenilline M (1) and with the formation of a double bond ∆ 10 in zosteropenilline R (9).Dehydration of i-7 is realized with the formation of a double bond ∆ 12 in the intermediate i-8, followed by oxidation of C-15 and shortening of the side chain, which gives 3, and oxidation of C-8 to form 2. The epoxidation of the double bond leads to i-12.The action of epoxide hydrolase on i-12 and the subsequent halogenation of tetraol i-13 yields zosteropenilline S (10).
At present time, selective estrogen receptor downregulators (SERDs) that act as pure antagonists by interfering with the binding of estradiol to estrogen receptors (ERs) are used for hormone receptor-positive (HR+) breast cancer treatment [32].Fulvestrant is a firstgeneration steroidal SERD approved by the FDA in 2007 for the treatment of ER-positive metastatic breast cancer [33].Selective estrogen receptor modulators (SERMs) modulate ER activity by changing the coregulators to which they bind, and nonsteroidal SERMs can be classified on the basis of their structures, such as triphenylethylenes (tamoxifen, toremifene and idoxifene), phenylindoles (bazedoxifene and pipindoxifene), benzothiophenes (raloxifene and arzoxifene), benzopyrans (acolbifene), tetrahydronaphthalenes (lasofoxifene) and dihydrobenzoxathiins [34].The sources of the reported SERDs and SERMs are plants, fungi and synthetic analogs of natural compounds, including xenoestrogens, which are structurally different from known estrogenic compounds.Examples of this include the p-hydroxyalkyl benzene derivative actinopolymorphol A from the actinomycete Actinopolymorpha rutilus [35] and the decalin derivatives fusarielins A, F, G and H from the fungus Fusarium graminearum [36].In the present study, we propose 1-acetylpallidopenilline A as a new mycoestrogen with potent antimetastatic activity against MCF-7 breast cancer cells.
On the other hand, the new possibility of 1-acetylpallidopenilline A was found to increase the cytotoxicity of doxorubicin.Other earlier decalin polyketides zosteropenillines A, C and G were found to be upregulators of autophagy-related cargo protein p62 (which increased levels associated with the inhibition of autophagy flux) in PC-3 cells [18].
It is obvious that the presence of a 1-acetyl chain in the structure of pallidopenillines leads to an increase in the antitumor properties of the compounds of these series.It is quite difficult to draw other conclusions about the influence of the structure on cytotoxic activity, since the differences in activity are small.Moreover, the investigation of the fungal Penicillium yezoense KMM 4679 strain resulted in the discovery of the decalin polyketides zosteropenillines O, К and S with protective effects against hypoxia-induced HEK-293 cell damage and induced mitochondrial lipid peroxide oxidation.Previously, it was shown that zosteropenillines B, H and J could downregulate NO levels in LPS-stimulated RAW264.7 murine macrophages, indicating their possible anti-inflammatory activity [18].New data on the antioxidant activity of zosteropillines may be interesting for future investigations.At present time, selective estrogen receptor downregulators (SERDs) that act as pure antagonists by interfering with the binding of estradiol to estrogen receptors (ERs) are used for hormone receptor-positive (HR+) breast cancer treatment [32].Fulvestrant is a first-generation steroidal SERD approved by the FDA in 2007 for the treatment of ER-

Cultivation of P. yezoense KMM 4679
The fungus was grown as described in [39].

Extraction and Isolation
At the end of the incubation period, the mycelia together with the medium was extracted with EtOAc (5 L).The obtained extract was dried in vacuo.The residue was dissolved in H 2 O−EtOH (4:1) (300 mL) and was extracted with n-hexane (0.2 L × 3) and EtOAc (0.2 L × 3).After evaporation of the EtOAc layer, the residual material (8.74 g) was passed over a Si gel, which was eluted followed by a step gradient from 100% n-hexane to 100% EtOAc (total volume 50 L).Fractions of 15 mL were collected and combined based on TLC analysis (Si gel, toluene-isopropanol, 6:1 and 3:1, v/v).
The n-hexane-EtOAc   1 and 2 S17).plates were dried for 2 h.Then, 100 µL/well of DMSO was added to each well to dissolve formazan crystals, and the absorbance was measured using a plate reader according to the manufacture's protocol.The results were reported as percentage inhibition normalized to the untreated control.The antibiotic gentamicin and antifungal agent nitrofungin were used as positive controls at 1 mg/mL; 1% DMSO in PBS served as a negative control.Examination was performed twice and in triplicate.

Colony Formation Assay
The influence of compounds on colony formation by PC-3 cells was tested by the clonogenic assay [41].The concentration of PC-3 cells was 0.33 × 10 3 /mL.The cells were incubated for 10 days, and then they were fixed with methanol (25 min), staining with 0.5% solution of crystal violet (25 min) and washing with PBS.The counting of grown colonies was carried out using a BIO-PRINT-Cx4 Edge-Fixed Pad-Container (Vilber, Collegien, France) using Bio-Vision Software user and service manual-v18.01 (Vilber, Collegien, France).The results are presented as colony inhibition in comparison with the control.

Migration Assay
Silicone 2-well inserts (Ibidi ® , Gräfelfing, Germany) were used for formation of a free cell zone in the PC-3 cells' monolayer.After removing the insert, the gap between the cells was 500 ± 50 µm.The cells were washed twice with PBS after removing the inserts and staining with (5,6)-carboxyfluorescein succinimidyl ester (CFDA SE) (Lumiprobe, Moscow, Russia) fluorescence dye.Then, cells were treated with compound or culture medium only (vehicle control) for 24 h.Cell migration into the wound area was observed under a fluorescence microscope (MIB-2-FL; LOMO, St. Petersburg, Russia) with objective 10× magnification.

Drug Combination Study
Experiments were performed as previously reported [42].To study the synergistic cytotoxic effect of the compound in combination with the clinically used anticancer drug doxorubicin, the PC-3 cells (5 × 10 3 cells/well in 96-well plates) were simultaneously treated with compound and doxorubicin at different concentrations.After 24 h of incubation, the cell viability was determined using the MTT assay described above.

Hypoxia
The HEK-293 cells were exposed to cobalt chloride (II) at a 500 µM for 1 h and then the compounds were added.After 24 h, the viability of the cells was measured via MTT assay as described earlier.

Lipid Peroxidation Level
The fluorescence probe MitoCLox (Lumiprobe, Moscow, Russia) was used for the detection of the lipid peroxidation level in CoCl 2 -treated cells.It was added in the cells for 1 h at a concentration of 200 nM.After 1 h, the cells were washed with PBS and fluorescence was measured in ratiometric mode with λem = 520/590 nm using a PHERAStar FS plate reader (BMG Labtech, Offenburg, Germany).The data were processed with MARS Data Analysis v. 3.01R2 (BMG Labtech, Offenburg, Germany) and calculated as a 520/590 ratio.

Statistical Data Evaluation
All the data were obtained in three independent replicates, and the calculated values are expressed as the mean ± standard error of the mean (SEM).A Student's t-test was performed using SigmaPlot 14.0 (Systat Software Inc., San Jose, CA, USA) to determine the statistical significance.The differences were considered statistically significant at p < 0.05.

Quantum Chemical Modeling
The B3LYP exchange-correlation functional, the polarization continuum model (PCM) and 6-311G(d) basis set implemented in the Gaussian 16 package of programs were used for all quantum chemical calculations [43].For compounds 1 and 3, conformational analysis was performed, and the significant (most stable) conformations were selected for further modeling of ECD spectra.
The statistical weights (g im ) of individual conformations were calculated according to the following equation: where ∆G im = G i − G m are the relative Gibbs free energies and index "m" denotes the most stable conformation.The ECD spectra were calculated using time-dependent density functional theory (TDDFT), the B3LYP functional, the PCM model and the 6-311G(d) basis set for conformations, of which relative Gibbs free energies satisfied to relation ∆G im ≤ 4 kcal/mol.Gauss-type functions were used for simulating the individual bands in theoretical spectra.The bandwidths ζ = 0.36 eV for 1 and ζ = 0.24 eV for 3 were used.The UV shifts ∆λ = +4 nm were used to obtain the best correspondence between the experimental and calculated spectra for 1 and 3.
The scaled theoretical and experimental ECD spectra were obtained according to the following equation: ∆ε sc (λ) = ∆ε(λ) ∆ε(λ peak ) where the denominator |∆ε(λ peak) | is a modulo of the peak value for the positive characteristic band at λ ≈ 290 nm in the corresponding ECD spectrum.

Conclusions
Ten new polyketides, named zosteropenilline M (1), 11-epi-8-hydroxyzosteropenilline M (2), zosteropenilline N (3), 8-hydroxyzosteropenilline G (4), zosteropenilline O (5), zosteropenilline P (6), zosteropenilline Q (7), 13-dehydroxypallidopenilline A (8), zosteropenilline R (9) and zosteropenilline S (10), were isolated from the ethyl acetate extract of the marine-derived fungus Penicillium yezoense KMM 4679.The absolute configurations of 1 and 3 were determined by time-dependent density functional theory (TD-DFT) calculations of the ECD spectra.The absolute configurations of 7, 9 and 10 were determined by a combination of the modified Mosher's method and ROESY data together with biogenetic relationships.A biogenetic pathway for 1−14 was proposed.Zosteropenillines O, К and S diminished cobalt chloride (II)-induced HEK-293 cell damage and recovered the high lipid peroxide oxidation level in the mitochondria of these cells.1-Acetylpallidopenilline A exhibited strong inhibition of human breast cancer MCF-7 cell colony formation and may be interesting for future studies as a new estrogen receptor modulator.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/md22070317/s1,Table S1.The strains of the species used in multi-locus phylogenetic analysis and GenBank accession numbers; Figures S1, S9

Figure 2 .
Figure 2. ML tree based on concatenated ITS-BenA-CaM-RPB2 sequences showing the phylogenetic position of the strain KMM 4679 among members of the genus Penicillium section Aspergilloides series Thomiorum.Bootstrap values (%) of 1000 replications with confidence values greater than 50% are indicated in the nodes.The scale bars represent 0.10 nucleotide substitutions per site.

Figure 2 .
Figure 2. ML tree based on concatenated ITS-BenA-CaM-RPB2 sequences showing the phylogenetic position of the strain KMM 4679 among members of the genus Penicillium section Aspergilloides series Thomiorum.Bootstrap values (%) of 1000 replications with confidence values greater than 50% are indicated in the nodes.The scale bars represent 0.10 nucleotide substitutions per site.

Figure 2 .
Figure 2. ML tree based on concatenated ITS-BenA-CaM-RPB2 sequences showing the phylogenetic position of the strain KMM 4679 among members of the genus Penicillium section Aspergilloides series Thomiorum.Bootstrap values (%) of 1000 replications with confidence values greater than 50% are indicated in the nodes.The scale bars represent 0.10 nucleotide substitutions per site.

Figure 11 .
Figure 11.Effect of 14 on the formation of PC-3 (a), HeLa (b) and MCF-7 (c) colonies.Data are presented as the mean ± SEM. * indicates the significant differences from the control with p-value ≤ 0.05.

Figure 11 .
Figure 11.Effect of 14 on the formation of PC-3 (a), HeLa (b) and MCF-7 (c) colonies.Data are presented as the mean ± SEM. * indicates the significant differences from the control with p-value ≤ 0.05.

Figure 12 .
Figure 12.The influence of 1-acetylpallidopenilline A (14) on the migration of HeLa (A) and MCF-7 (B) cells.Data are presented as the mean ± SEM. * indicates the significant differences from the control with p-value ≤ 0.05.

Figure 12 .
Figure 12.The influence of 1-acetylpallidopenilline A (14) on the migration of HeLa (A) and MCF-7 (B) cells.Data are presented as the mean ± SEM. * indicates the significant differences from the control with p-value ≤ 0.05.

Figure 12 .
Figure 12.The influence of 1-acetylpallidopenilline A (14) on the migration of HeLa (A) and MCF-7 (B) cells.Data are presented as the mean ± SEM. * indicates the significant differences from the control with p-value ≤ 0.05.

Figure 13 .
Figure 13.Combined effect of 14 and doxorubicin on the viability of PC-3, HeLa and MCF-7 cells.Data are presented as the mean ± SEM. * p-value ≤ 0.05 considered significant.

Figure 13 .
Figure 13.Combined effect of 14 and doxorubicin on the viability of PC-3, HeLa and MCF-7 cells.Data are presented as the mean ± SEM.

Figure 14 .
Figure 14.The influence of 14 (at a 75 µ M) on cell viability in the presence of 4-OHT (at 10 µ M).Data are presented as the mean ± SEM. * p-value ≤ 0.05 considered significant.

Figure 14 .
Figure 14.The influence of 14 (at a 75 µM) on cell viability in the presence of 4-OHT (at 10 µM).Data are presented as the mean ± SEM. * p-value ≤ 0.05 considered significant.
* Chemical shifts were measured at 700 MHz in CDCl 3 .
1The concentration of each compound was 100 µM.